CN111088368B - Blood mRNA detection kit and detection method - Google Patents
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Abstract
The invention discloses a blood mRNA detection kit and a detection method, which comprise 9 pairs of specific multiplex PCR primer pairs shown in SEQ ID NO. 1-18 and 16 SNP single-base extension primers shown in SEQ ID NO. 19-34, are used for detecting 16 SNP molecular markers on 5 blood specific mRNA molecules, can complete the operation of detecting blood RNA with the ratio as low as 1% or the content as low as 0.1ng from complex mixed spots, accurately separate blood from other body fluids and skin tissues, and quickly and accurately trace to the source and locate a suspect.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a blood detection kit and a detection method.
Background
In forensic work, complex mixed spots are one of the most common biological examination materials and are always the key and difficult points of examination. Complex mixed spots include: (1) mixed plaques consisting of multiple body fluids of multiple persons; (2) the composition ratio of the mixed spots is extremely unbalanced (one component composition ratio is less than 10%). The mainstream technology for analyzing the mixed spots formed by a plurality of people and a plurality of body fluids at home and abroad is to type STRs based on a single cell separation technology, capillary electrophoresis or a second-generation sequencing platform.
Some mRNA molecules in blood have body fluid and tissue specificity, and the specific mRNA can be expressed and detected only in blood, while other body fluids do not express. At present, specific mRNA molecules cannot be singly utilized to identify individuals in forensic medicine, and the method is only mainly used for deducing body fluid spot type identification and combining mRNA and DNA co-extraction technology to realize spot identification and individual identification. However, the combination of mRNA molecules and DNA achieves the purpose of identifying the type of body fluid spots and individual recognition, but does not solve the problem of complex mixed spots.
Although the second generation sequencing platform has good analysis capability on simple mixed spots, when complex mixed spots are analyzed, the second generation sequencing platform has obstacles which are difficult to overcome in the aspects of sequencing fragment splicing, sequencing error, detection of components with small proportion to unbalanced mixed spots and the like. The traditional analysis method can also have the events of loss of alleles, appearance of new alleles, shadow bands and the like, and the condition that a plurality of individuals share the same alleles can be met during analysis, so that the interpretation of an identification result is difficult, and subjective deviation and even errors occur. For mixed spots constituting a specific imbalance, it is difficult to find the cell components of the secondary donor (< 10%), resulting in a bottleneck (5-10%) in the detection sensitivity of complex mixed spots that cannot always be broken through.
In addition, the traditional method can only compare the analysis result in the mixed spot with the suspect, so that the suspect is difficult to directly lock by using the blood information in the mixed spot, and the method is a big problem aiming at the research of the complex mixed spot at present.
Disclosure of Invention
The invention aims to provide a blood mRNA detection kit and a detection method, which are used for identifying a blood sample with RNA content as low as 1% or 0.1ng RNA from a complex mixed spot and realizing the active search of a suspect.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the reported blood specific mRNA molecules are selected, and the database is used to screen SNP molecular markers with good polymorphism (0.1 < MAF < 0.5), wherein 5 specific genes from blood corresponding to SNP are shown in Table 1.
The blood mRNA detection kit comprises 9 pairs of specific multiplex PCR primer pairs shown in SEQ ID NO. 1-18 and 16 SNP single base extension primers shown in SEQ ID NO. 19-34, and is used for detecting any one or more of the following 16 SNP molecular markers:
rs3753058、rs3753059、rs1793174、rs2304871、rs17121881、rs229586、rs229587、rs77806、rs1626923、rs1741487、rs184528、rs1741488、rs7682260、rs7687256、rs7658293、rs4867。
wherein, the PCR primer pair of rs3753058 and rs3753059 is an upstream primer shown in a sequence SEQ ID NO.1 and a downstream primer shown in a sequence SEQ ID NO. 2.
The PCR primer pair of rs1793174 is an upstream primer shown in a sequence SEQ ID NO.3 and a downstream primer shown in a sequence SEQ ID NO. 4.
The PCR primer pair of rs2304871 is an upstream primer shown as a sequence SEQ ID NO.5 and a downstream primer shown as a sequence SEQ ID NO. 6.
The PCR primer pair of rs17121881 is an upstream primer shown in a sequence SEQ ID NO.7 and a downstream primer shown in a sequence SEQ ID NO. 8.
The PCR primer pair of rs229586 and rs229587 is an upstream primer shown as a sequence SEQ ID NO.9 and a downstream primer shown as a sequence SEQ ID NO. 10.
The PCR primer pair of rs77806 is an upstream primer shown as a sequence SEQ ID NO.11 and a downstream primer shown as a sequence SEQ ID NO. 12.
The PCR primer pair of rs1626923 is an upstream primer shown in sequence SEQ ID NO.13 and a downstream primer shown in sequence SEQ ID NO. 14.
The PCR primer pair of rs1741487, rs184528 and rs1741488 is the upstream primer shown in the sequence SEQ ID NO.15 and the downstream primer shown in the sequence SEQ ID NO. 16.
The PCR primer pair of rs7682260, rs7687256, rs7658293 and rs4867 is an upstream primer shown by a sequence SEQ ID NO.17 and a downstream primer shown by a sequence SEQ ID NO. 18.
The SNP single-base extension primers shown in SEQ ID NO. 19-34 are designed aiming at the 16 SNP molecular markers respectively, and the corresponding modes are as follows:
the sequence of the extension primer corresponding to rs3753058 is SEQ ID NO. 19. The sequence of the extension primer corresponding to rs3753059 is SEQ ID NO. 20. The sequence of the extension primer corresponding to rs1793174 is SEQ ID NO. 21. The sequence of the extension primer corresponding to rs2304871 is SEQ ID NO. 22. The sequence of the extension primer corresponding to rs17121881 is SEQ ID NO. 23. The sequence of the extension primer corresponding to rs229587 is SEQ ID NO. 24. The sequence of the extension primer corresponding to rs77806 is SEQ ID NO. 25. The sequence of the extension primer corresponding to rs1626923 is SEQ ID NO. 26. The sequence of the extension primer corresponding to rs1741487 is SEQ ID No. 27. The sequence of the extension primer corresponding to rs184528 is SEQ ID NO. 28. The sequence of the extension primer corresponding to rs1741488 is SEQ ID No. 29. The sequence of the extension primer corresponding to rs7682260 is SEQ ID NO. 30. The sequence of the extension primer corresponding to rs7687256 is SEQ ID NO. 31. The sequence of the extension primer corresponding to rs7658293 is SEQ ID NO. 32. The sequence of the extension primer corresponding to rs4867 is SEQ ID NO. 33. The sequence of the extension primer corresponding to rs229586 is SEQ ID NO. 34.
Further, the invention also provides a detection method of the blood mRNA detection kit, which comprises the following steps: firstly, extracting total RNA of a sample, carrying out reverse transcription to obtain cDNA, and adding a multiplex PCR amplification system. Then, the amplification product is purified and added into a single-base extension system, and the extension product is purified. Finally using capillary electrophoresis, ABI®3130 DNA sequencer and ABI®The GeneMapper ID V3.2 analysis detected extension products.
Compared with the prior art, the invention has the following beneficial effects:
the invention can not only judge whether blood exists in the mixed spot, but also realize accurate tracing to blood donors in the mixed spot, the extremely unbalanced mixed spot sample and the old spot which are formed by a plurality of human fluids, and is not interfered by other body fluid donors. Complete typing results for blood polymorphisms were obtained when the sample RNA amount was as low as 0.1ng or the total RNA content was as low as 1%. The perfect separation of blood and other types of body fluids and tissues in the complex mixed spots can be realized, the suspect can be found by directly utilizing the analysis result, and the suspect can be actively found after the database is built.
Drawings
FIG. 1 is a diagram of standard typing of 16 SNPs on blood-specific mRNA molecules.
FIG. 2 is the result of selecting 8 SNP molecular markers by Sanger sequencing.
FIG. 3 shows the SNP typing results of blood samples and simulation cases, wherein (A) is the primary typing result of blood sample SNP, and (B) is the typing result of complex spot SNP containing 1% blood RNA in simulation cases.
FIG. 4 shows the SNP typing results of blood spot samples stored at room temperature for 6 months.
FIG. 5 shows the results of amplification of a template containing 0.1ng of blood RNA and SNP typing.
FIG. 6 shows the SNP typing results of human body fluids from different sources after RNA extraction, wherein (A) is saliva, (B) is seminal plaque, (C) is vaginal secretion, (D) is skin tissue, and (E) is menstrual blood.
Detailed Description
The following examples further describe embodiments of the present invention. The following examples are only for illustrating the technical solutions of the present invention more clearly, and do not limit the scope of the present invention. Various changes, modifications, substitutions and alterations to these embodiments will be apparent to those skilled in the art without departing from the principles and spirit of this invention.
Example 1, screening of mixed plaques.
1. And (4) preparing a template.
Whole blood RNA was extracted using the RNAioso Plus kit (Takara). After quantification, 100ng of RNA was used as a template for Reverse Transcription using the GoScript Reverse Transcription Kit (Promega) Kit. Obtaining a template cDNA.
2. Performing composite amplification and purification.
1) The template cDNA representing 5ng of RNA was added to a 10. mu.L reaction: 5 μ L of 2 × Mastermix; mu.L of 10 XPrimer Mix (Primer concentrations are given in Table 2) and the reaction was made up to 10. mu.L with water. PCR thermal cycling conditions were as follows: at 95 ℃ for 10 min; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 20 s; 5min at 72 ℃.
2) And a composite amplification product purification system: mu.L of the amplification product, 1.3U SAP (shrimp alkaline phosphatase), 6U Exo I (exonuclease I), and water were added to make up the reaction system to 10. mu.L. And (3) purification conditions: 1h at 37 ℃; 95 ℃ for 15 min.
3. SNP single base extension reaction and purification.
1) An extension system: mu.L of PCR-purified product, 5. mu.L of SNaPshot Mix, 1. mu.L of extension primer (primer concentrations see Table 2), extension conditions: 1min at 96 ℃; 35 cycles of 96 ℃ for 10s, 50 ℃ for 5s and 60 ℃ for 30 s; 60 ℃ for 1 min.
2) And a purification system: mu.L of single base extension product, 1U SAP (shrimp alkaline phosphatase), water supplemented to 5. mu.L. And (3) purification conditions: 1h at 37 ℃; 95 ℃ for 15 min.
4. Capillary electrophoresis and analysis.
1) And a denaturation treatment system before electrophoresis: 1.5. mu.L of purified product, 0.1. mu.L of internal standard (GeneScan-LIZ 120), 10. mu.L of formamide (Hi-Di formamide); the treatment conditions are as follows: 5min at 95 ℃ and 3min at 0 ℃.
2) By ABI®3130 the DNA sequencer detects the extension primers and analyzes the extension products by ABI®GeneMapper ID V3.2. FIG. 1 shows the results of standard typing of 16 SNPs on blood-specific mRNA molecules. The sequence numbers and SNP mapping are shown in Table 1. Sanger sequencing is carried out on 8 SNP molecular markers, and as shown in figure 2, the typing result is consistent with that of the kit.
5. Complex mixed plaques containing 1% blood RNA were detected.
The results are shown in FIG. 3, wherein (A) shows the original typing results of blood samples, and (B) shows the typing results of complex mixed spots using the kit. The two types are consistent, which shows that the method can detect a very small amount of blood in the mixed spots and is not interfered by other body fluids.
Example 2, screening of old spots.
The blood spot sample preserved for 6 months at room temperature is used as a template for detection by using the kit, and the result is shown in figure 4, so that all typing results are obtained.
Example 3 screening for very unbalanced mixed spots.
The amplification and SNaPshot typing of the template containing 0.1ng of RNA was carried out using the kit, and the results are shown in FIG. 5, and all typing results were also obtained.
Example 4 screening of other body fluids.
The kit is used for detecting saliva, seminal plaques, vaginal secretion, skin tissues and menstrual blood, the result is shown in figure 6, and the 16 SNP molecular markers selected by the invention are not detected in other body fluids.
SEQUENCE LISTING
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Claims (6)
1. A blood mRNA detection kit comprises 9 pairs of specific multiplex PCR primer pairs shown in SEQ ID NO. 1-18 and 16 SNP single base extension primers shown in SEQ ID NO. 19-34, and is used for detecting the following 16 SNP molecular markers:
rs3753058、rs3753059、rs1793174、rs2304871、rs17121881、rs229587、rs77806、rs1626923、rs1741487、rs184528、rs1741488、rs7682260、rs7687256、rs7658293、rs4867、rs229586。
2. the detection kit according to claim 1, wherein the 9 pairs of specific multiplex PCR primers shown in SEQ ID No. 1-18 respectively amplify the following SNP molecular markers:
the PCR primer pair of rs3753058 and rs3753059 is an upstream primer shown in a sequence SEQ ID NO.1 and a downstream primer shown in a sequence SEQ ID NO. 2;
the PCR primer pair of rs1793174 is an upstream primer shown by a sequence SEQ ID NO.3 and a downstream primer shown by a sequence SEQ ID NO. 4;
the PCR primer pair of rs2304871 is an upstream primer shown as a sequence SEQ ID NO.5 and a downstream primer shown as a sequence SEQ ID NO. 6;
the PCR primer pair of rs17121881 is an upstream primer shown by a sequence SEQ ID NO.7 and a downstream primer shown by a sequence SEQ ID NO. 8;
the PCR primer pair of rs229586 and rs229587 is an upstream primer shown as a sequence SEQ ID NO.9 and a downstream primer shown as a sequence SEQ ID NO. 10;
the PCR primer pair of rs77806 is an upstream primer shown as a sequence SEQ ID NO.11 and a downstream primer shown as a sequence SEQ ID NO. 12;
the PCR primer pair of rs1626923 is an upstream primer shown by a sequence SEQ ID NO.13 and a downstream primer shown by a sequence SEQ ID NO. 14;
the PCR primer pair of rs1741487, rs184528 and rs1741488 is an upstream primer shown in a sequence SEQ ID NO.15 and a downstream primer shown in a sequence SEQ ID NO. 16;
the PCR primer pair of rs7682260, rs7687256, rs7658293 and rs4867 is an upstream primer shown by a sequence SEQ ID NO.17 and a downstream primer shown by a sequence SEQ ID NO. 18.
3. The detection kit according to claim 1, wherein the 16 SNP single-base extension primers shown by SEQ ID numbers 19-34 are respectively extended with the following SNP molecular markers:
the sequence of the extension primer corresponding to rs3753058 is SEQ ID NO. 19; the sequence of the extension primer corresponding to rs3753059 is SEQ ID NO. 20; the sequence of the extension primer corresponding to rs1793174 is SEQ ID NO. 21; the sequence of the extension primer corresponding to rs2304871 is SEQ ID NO. 22; the sequence of the extension primer corresponding to rs17121881 is SEQ ID NO. 23; the sequence of the extension primer corresponding to rs229587 is SEQ ID NO. 24; the sequence of the extension primer corresponding to rs77806 is SEQ ID NO. 25; the sequence of the extension primer corresponding to rs1626923 is SEQ ID NO. 26; the sequence of the extension primer corresponding to rs1741487 is SEQ ID No. 27; the sequence of the extension primer corresponding to rs184528 is SEQ ID NO. 28; the sequence of the extension primer corresponding to rs1741488 is SEQ ID No. 29; the sequence of the extension primer corresponding to rs7682260 is SEQ ID NO. 30; the sequence of the extension primer corresponding to rs7687256 is SEQ ID NO. 31; the sequence of the extension primer corresponding to rs7658293 is SEQ ID NO. 32; the sequence of the extension primer corresponding to rs4867 is SEQ ID NO. 33; the sequence of the extension primer corresponding to rs229586 is SEQ ID NO. 34.
4. The test kit according to claim 1, wherein the amplification product of each pair of specific multiplex PCR primers comprises at least 1 SNP on a blood-specific mRNA molecule and at most 4 SNPs.
5. The detection kit according to claim 1, wherein the 16 SNP molecular markers on the mRNA are derived from 5 blood-specific genes: SPTB, GYPA, CD3G, ANK1, AMICA 1.
6. The method for detecting a non-disease diagnosis using the detection kit according to claim 1, comprising the steps of:
1) extracting RNA of a sample to be detected as a template, carrying out reverse transcription on the RNA to be detected to obtain cDNA, and carrying out multiple PCR by using 9 pairs of specific multiple PCR primer pairs shown in SEQ ID NO. 1-18 to obtain an amplification product;
2) purifying the amplification product, and obtaining an extension product through a single base extension system by using 16 SNP single base extension primers shown in SEQ ID NO. 19-34;
3) and purifying the extension product, and analyzing by using capillary electrophoresis, a DNA sequencer and gene analysis software.
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| CN105658818A (en) * | 2013-10-21 | 2016-06-08 | 泰华制药工业有限公司 | Genetic markers predictive of response to glatiramer acetate |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105658818A (en) * | 2013-10-21 | 2016-06-08 | 泰华制药工业有限公司 | Genetic markers predictive of response to glatiramer acetate |
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| Title |
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| Candidate Malaria Susceptibility/Protective SNPs in Hospital and Population-Based Studies: The Effect of Sub-Structuring;Nahid A Eid等;《Malaria Journal》;20100531;第9卷(第1期);119 * |
| KCNQ1 Polymorphism and Susceptibility to Sudden Cardiac Death in Chinese Population;Gengqian Zhang等;《Elsevier Editorial System(tm) for The Lancet Manuscript Draft》;20181128;1-10 * |
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